Alzheimer's disease (AD) is a progressive neurodegenerative disease likely caused by a combination of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (apo) E4, is of significant importance as apoeE4 carriers account for 65-80 percent of all AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. Emerging data suggests that apoE4 is sensitive to proteolytic cleavage and thus contributes to the underlying molecular pathology associated with AD possibly through a loss of function. However, the molecular mechanisms underlying the proteolytic cleavage of apoE4, including the identity of the protease involve, has not been clarified. The purpose of this study is to identify the protease responsible for ApoE4 cleavage and to determine whether or not this cleavage event occurs in the AD brain. Using a site-directed antibody to cleaved apoE4 we have recently determined a major cleavage fragment of apoE4 of 18 KDa is present in recombinant forms following purification of apoE4 from E. coli and is present in situ in AD brain sections. We hypothesize that this proteolytic event represents a novel pathway for apoE4 cleavage and that caspases are responsible for this cleavage in the AD brain. Experiments described in Aim 1 will rigorously test the hypothesis that apoE4 is cleaved by caspases to generate a 18 kDa amino-terminal fragment. These experiments will characterize the cleavage event by analyzing the production of the apoE4 fragment following incubation of purified apoE4 or recombinant forms with caspase-3 in a cell-free system or following caspase activation in a model system of apoptosis. These experiments will be aided through the development of a site-directed cleavage antibody that specifically detects the amino-terminal 18 kDa fragment of apoE4 following cleavage at D172. Experiments outlined in Aim 2 will determine whether proteolytic cleavage is unique to only the E4 isoform, and not other known isoforms including apoE2 or apoE3. We hypothesize that only apoE4 will be uniquely sensitive to proteolytic cleavage compared to other isoforms and thus, this will serve as an underlying event connecting apoE4 proteolysis to an enhanced risk of late-onset AD. Using our site-directed cleavage antibodies, experiments in Aim 3 will determine if this same apoE4 fragment is generated in the AD brain and if so, what cell type. Due to the role of caspases in cleaving tau and contributing to the formation of neurofibrillary tangles, we hypothesize this 18 kDa fragment of apoE4 will localize within neurofibrillary tangles of the AD brain. Both immunohistochemical analysis using fixed post mortem brain sections as well as Western blot analysis from AD samples will be performed and compared to age-matched controls.